Cell Culturing and Fermentation

Biotechnology II

Cell Culture

Definition: the in vitro growth of cells isolated from multi-cellular organisms

Process: Cells will continue dividing until they fill up the container; cell to cell contact stops cell division

Uses: vaccines, research of all kinds including stem cell, recombinant DNA, production of antibodies

Types of Cell used

Bacterial cells were first used to make genetically engineered products and they reproduce very quickly. Being prokaryotes makes them simple

Yeast cells are eukaryotic but still fairly simple to use

Plant cells are found within tissue, more difficult to use, slow growing

Insect cells are closer to humans but not as fragile as mammalian cells

Mammalian cells are commonly used but they do grow slower and are more fragile than bacterial cells. Also have more complex nutrient requirements

Cell Culture Process

Contaminating the cell culture is area of concern To avoid contamination mfg. techs do the following:

Sterilize all containers used Change the media frequently

Media is the source of nutrition, pH indicator often times included in media to help monitor pH which is critical to cell survival

The media used depends on the type of cells to culture

Equipment Required

CO2 incubator: for proper atmospheric conditions

Laminar flow hood: for sterility

Hemocytometer: for cell counting

Inverted Microscope: for viewing cells on hemocytometer

Cell Culture Process

Count cells Using a hemocytometer the cells are counted to

determine if they are ready to harvest Can stain cells with trypan blue to determine if

they are still alive. All cells which exclude the dye are viable. All stained cells are dead.

Cell Culture Process

View cells Inverted microscope is used to

determine cell growth and activity Adherent cells are ones which are

stuck to the flask or vessel

Cell counting video for cell culture

Fermentation: Yeast on the Rise

Basic Reaction Dextrose (glucose) = alcohol + CO2

If using maltose as your sugar it first is converted to dextrose and then is broken down.

Quantifying alcohol or CO2 production are two standard approaches to measuring yeast fermentation.

Biofuel Ethanol is referred to as a biofuel as it is

manufactured by fermenting grains, plant biomass, or other microorganisms such as yeast.

Can be used directly as fuel although most often mixed with gasoline (gasohol).

Billions of gallons of ethanol are produced each year

Bioethics of Biofuels Should grains be used as an energy source? Each bushel (56 lbs.) yields 2.5 gallons Amt. of farmed land has declined and population

has grown 50% over last 25 years. 6% of US corn crop is used for ethanol production There are environmental benefits of ethanol compared to petroleum

Basics of apparatus to measure CO2

Fermentation reaction in a vessel (flask or syringe) Need to measure volume displaced from CO2 production.

Could be pipet, graduated cylinder, syringe

Chemostat Design Sketches

Fermentation reaction

Water in pipet

Fermentation reaction

Water in cylinder

Record starting volume of water and measure displaced over time

Formulation Components Sugar source: glucose, sucrose, maltose, lactose Water: approx. 10mL/g of glucose Yeast: approx. 8g/100 mL

Could make up as a solution (add 1:1 w/sugar soln.)

Could add dry powder (approx. 0.8gm/10mL)

Experimental Design

Need to modify different variables to determine optimum formulation Temperature Type of sugar Type of yeast Qty of sugar and yeast pH

Experimental Design

Need to run controlled experiments Ensure apparatus is working consistently Record all data Only modify one variable at a time Analyze data and determine next experiment

Data Analysis

Plot data on graph paper Record mL of CO2 on y axis, time on x axis

0 min 0.1 mL

2 min 0.3 mL

4 min 2.0 mL

5 min 4.0 mL time

Find the straight part of the curve and calculate the rate of CO2/min

Fermentation and Cellular respiration

Fermentation yields energy via cellular respiration What is cellular respiration?

It requires O2

Glycolysis Krebs cycle Electron transport

Cellular Respiration

So what is fermentation?

An enzymatic process that allows cells to obtain energy from carbs even in the absence of oxygen.

If O2 is not present cellular respiration is shut down Glycolysis still occurs if pyruvate is diverted to the

fermentation pathway (NADH converted to NAD)

Which is better w/without O2?

Go back to Candy bar picture.

So, how do yeast cells live in a fermenter?

It sounds like there is too little ATP to keep us alive in an anaerobic environment?

For higher organisms this is true. But what do you know about yeast?

It is prokaryotic and uses a small amount energy Yeast can import and metabolize carbs very rapidly which

compensates for small production of ATP Because yeast metabolizes carbs so quickly it is a great generator of

CO2 and that is why it is commonly used!

Report and Graphs

Final report should include the following: Hypothesis Procedure Results Discussion Conclusion Questions Cited References

Report and Graphs

Graph some of your data such as the difference in fermentation rates between the different sugars, yeast, temp., etc.

Data table of time and mL displayed for each experiment Could graph fermentation rate vs. sugar concentration